Registration Dossier

Administrative data

Description of key information

skin irritation (OECD 439): not irritating
eye irritation (MatTek’s EpiOcular (ET50) protocol): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 - 22 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ (Epi-200)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 19674 Kit B
- Delivery date: 17 Sep 2014
- Date of initiation of testing: 17 Sep 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile hood

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT was prepared on the day of testing using MTT-100 Assay Kit Components.
- Incubation time: 42 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 ± 1 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.5 h.
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
approximately 42 h
Number of replicates:
triplicates for each treatment and control group
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
101
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Other effects / acceptance of results:
OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.742, 1.808 and 1.904) was in the range of ≥ 0.8 and ≤ 2.8 for 60 minutes treatment, thus showing the quality of tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.3% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The standard deviations of the % variabilities of the test substance, the positive and negative controls were < 10%, thus ensuring the validity of the study.

Table 2. Results after treatment

 Dose Group

 Treatment Interval

Absorbance 570 nm Tissue 1*

Absorbance 570 nm Tissue 2*

Absorbance 570 nm Tissue 3*

Mean Absorbance of 3 Tissues 

Rel. Absorbance [%] Tissue 1, 2 + 3** 

Relative Standard Deviation [%] 

Mean Rel. Absorbance [% of Negative Control]*** 

Negative Control

 60 min

 1.742

 1.808

 1.904

 1.818

95.8; 99.5; 104.7

 4.5

 100.0

Positive Control

 60 min

 0.126

 0.117

 0.103

 0.115

6.9; 6.4; 5.7

 9.7

 6.3

Test Item

 60 min

 1.889

 1.740

 1.877

 1.835

103.9; 95.7; 103.2

4.5 

101.0 

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]: (100 x (absorbance tissue)) / (mean absorbance negative control))

*** relative absorbance per treatment group [rounded values]: (100 x mean absorbance test item/positive control) / (mean absorbance negative control)

 

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (101.0%; threshold for irritancy: ≤ 50%), consequently, the test item was not irritant to skin.

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
In the skin irritation study in vitro, the treatment with the test substance resulted in a cell viability of 101% and thus the test substance does not possess any skin irritating potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 - 19 Sep 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to MatTek’s EpiOcular (ET50) protocol, equivalent to MatTek’s EpiOcular (EIT) protocol under current (2014) ECVAM validation and GLP. Study was perfomed before release of OECD 492 in July 2015.
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
Jul 2015
Deviations:
yes
Remarks:
study was performed before publication of OECD 492; i.e. there are deviations in application of the test and control substance and in tissue viability measurements
Principles of method if other than guideline:
The study was performed according to MatTek’s EpiOcular (ET50) protocol, equivalent to MatTek’s EpiOcular (EIT) protocol under current ECVAM validation.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Test material information:
Composition 1
Species:
human
Strain:
other: EpiOcular™
Details on test animals or tissues and environmental conditions:
TEST EYE MODEL
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 19185

TEST METHOD
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The test consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

ADAPTATION TO CELL CULTURE CONDITIONS
At least 1 hour before dosing, EpiOcular™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into cell culture plates containing the pre-warmed assay medium. Additional cell cultures plates containing an adequate volume of medium were prepared as holding plates. The holding plates were pre-warmed in an incubator until use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
Vehicle:
unchanged (no vehicle)
Controls:
other: The negative control was deionised water and 0.3% Triton X-100 solution was used as positive control.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 100 µL

POSITIVE CONTROL SUBSTANCE
- Positive control substance: 0.3% Triton X-100 solution in deionised water
- Amount applied: 100 µL

NEGATIVE CONTROL SUBSTANCE
- Negative control substance: deionised water
- Amount applied: 100 µL
Duration of treatment / exposure:
Test substance: 3, 30 and 60 min
Positive control: 15 and 45 min
Negative control: 60 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
The test was performed in duplicates for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The tissues were gently rinsed with PBS to remove any residual test material.
- Time after start of exposure: 3, 30 and 60 min (test substance); 15 and 45 min (positive control); 60 min (negative control)
- Post-treatment incubation period: at least 10 minutes but not longer than 20 minutes

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed after the incubation period. Therefore, tissues were incubated in 300 µL freshly prepared MTT-reagent for 3 h at 37 ± 1.5 °C and 5 ± 0.5% CO2. After aspiration of the MTT reagent, tissues were rinsed three times with PBS. Extraction of the formazan product was carried out in 2 mL isopropanol for 18.5 h. The optical density was measured at 570 nm wave length in a microplate reader.
Irritation parameter:
other: rel. absorbance (% of negative control) / mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
112
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: rel. absorbance (% of negative control) / mean value of 2 tissues
Run / experiment:
30 min exposure
Value:
117.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: rel. absorbance (% of negative control) / mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
94.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritant / corrosive response data:
The relative absorbance values of the test item, corresponding to the cell viability, did not or did only irrelevantly decrease (94.8%) compared with the result of the negative control, consequently the test item was classified as non irritant.
Due to the lack of cytotoxicity, an ET50 value could not be calculated.

Table 2. Results after treatment with the test substance.

 

Treatment interval (min)

Mean Absorbance of
2 tissues*

Rel. Absorbance
(% of negative control)

Negative control

60

1.1600

100.0

Positive control

15

0.6920

59.7

45

0.3262

28.1

Test substance

3

1.2988

112.0

30

1.3678

117.9

60

1.0992

94.8

* Mean of three replicate wells after blank correction

ET50 of the test item = could not be calculated since viability was not reduced below 50%

ET50 of the positive control = 24.2 min

Optical evaluation of the MTT-reducing capacity of the test substance after 1 h incubation with MTT-reagent did not show blue colour.

Both acceptability criteria of the assay were met:

The corrected mean OD values of the two tissues exposed to the negative control were ≥ 0.8 (1.2024 and 1.1175).

The ET50-value of the positive control is determined during the exposure period ≤ 30 min (24.2 min). 

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the human cornea model test the test substance does not possess any eye irritating potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
There is only one study available.

Justification for selection of eye irritation endpoint:
There is only one study available.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.